INTRODUCTION:

Chemically brimonidine tartrate (Figure 1) is 5-bromo-6-(2-imidazolidinylideneamino) quinoxaline L-tartrate. It is an alpha-2 adrenergic agonist which is used for lowering intraocular pressure in patients with open-angle glaucoma or ocular hypertension^1-2. Literature survey of brimonidine tartrate revealed that, there exist a few reports on high performance thin layer chromatography². The drug is also explored for its estimation by using high performance liquid chromatography^3-5 and liquid chromatography-mass spectrometry (LC/MS/MS)⁶. There also exists a report on stability indicating study of the drug by using ultra performance liquid chromatography methods⁷. In the present work we developed a simple, reliable and precise RP-HPLC method for analysis of brimonidine tartrate. The developed method can be successfully used for analysis of brimonidine tartrate.

Fig.1: Structure of brimonidine tartrate

EXPERIMENTAL:

Drug and reagents:

Pure sample of brimonidine tartrate was obtained as gratis from Wockhardt Ltd. (Aurangabad, India) and it was used without further purification. HPLC grade methanol was procured from Merck specialties Pvt. Ltd. (Mumbai, India). Analytical reagents (AR) grade ortho phosphoric acid and potassium dihydrogen phosphate were procured from Qualigens fine chemicals Pvt. Ltd. (Mumbai, India). Ultra pure water obtained from Millipore water purification system (Molsheim, France) was used throughout the study.

Equipments:

UV-visible spectrophotometer (PerkinElmer, Shelton, CT, USA) was used for the determination of detection wavelength. A high-performance liquid chromatography (HPLC) system from PerkinElmer (Shelton, CT, USA) was used for the analysis of samples, which consisted of online degasser, sample injector (Rheodyne sample loop 20 μL), UV-visible detector (Series 200), pump (Reciprocating, series 200) and computer system loaded with Total Chrome Navigator (version 6.3.1) software. Other equipment used were, pH meter (Labindia, Mumbai, India), weighing balance (Shimadzu, AUX220, Kyoto, Japan) and a micro-pipette (Erba Biohit, Mannheim, Germany). All the samples were analyzed by using Kromasil C-18 column 250mm x 4.6mm i.d., particle size 5μm; (Eka Chemicals AB, Bohus, Sweden).

Preparation of sample for HPLC analysis:

Stock solution of 1000 μg mL^-1was prepared by dissolving 10 mg nicardipine in 10 mL methanol. Sample solution was prepared by dissolving stock solution mobile phase as per the required concentrations.

Selection of detection wavelength:

Appropriate dilutions of the brimonidine tartrate were prepared using water as diluent. Solution was scanned using double beam UV-visible spectrophotometer between the ranges of 400 to 200 nm. The detection wavelength was found to be 246 nm.

HPLC method development and validation:

Detection wavelength for the drug was found to be 246 nm and hence methanol was used as an organic phase. UV spectra of brimonidine tartrate is shown in Figure 2. Various trials were conducted to achieve adequate peak symmetry and response factor by varying the concentration of organic phase and pH of the buffer system. Finally methanol and buffer (phosphate buffer 10 mM) with pH 3.0 was selected to analyze brimonidine tartrate samples. The chromatogram of the drug is shown in Figure 3. The flow rate, injection volume and detection wavelength were 1 mL min^-1, 20 μL and 246 nm, respectively.

The developed method was validated with respect to various parameters outlined in the International Conference on Harmonization (ICH) guidelines Q2 (R1)⁸. A stock solution containing 1 mg mL^-1 drug was prepared in MeOH and the linearity was established by using the concentration in the range of 10-30 μg mL^-1. The solutions were prepared in triplicate and analyzed by injecting 20 μL into HPLC. The intra-day and inter-day precision were established by analyzing 10 μg mL^-1, 20 μg mL^-1 and 30 μg mL^-1 drug solutions three times on the same day and the next day, respectively. Accuracy was determined by spiking three known concentrations of the drug, viz., 80%, 100% and 120% in a 10 mg/mL of measured standard stock solution in triplicate and then determining the percent recovery of the added drug. Limit of detection and limit of quantitation were assessed by determining the signal to noise ratio of the injected concentration of the analyte. System suitability of the developed method was also determined by with respect to different parameters.

RESULT AND DISCUSSION:

Method development and validation:

An acceptable separation was achieved by using buffer of pH 3.0 along with the organic phase in a gradient mode shown in Table 1. The developed method was validated with respect to linearity, precision, accuracy, specificity and selectivity.

Fig.2: UV chromatogram of brimonidine tartrate

Table 1: Gradient program of mobile phase

Steps	Time	Flow	%A	%B
0	0.5	1.0	10	90
1	12.0	1.0	33	77
2	2.0	1.0	10	90
3	5.0	1.0	10	90

Fig.3: HPLC chromatogram of brimonidine tartrate

Linearity:

The response for the drug was found to be linear in the investigated concentration range. The values of slope and correlation coefficient (R²) were 227751 and 0.9994, respectively. The linearity data are shown in Table 2, Fig. 4.

Fig. 4: Linearity of brimonidine tartrate.

Table: 2 Linearity data for brimonidine tartrate.

Parameter	Brimonidine tartrate
Slope	32750.45
Correlation coefficient	0.9994
Intercept Slope of standard deviation	51633.56 10251.32

Precision:

Inter-day and intra-day precision data are enlisted in Table 3. The RSD values ranging from 0.98% to 1.44% for intra-day precision and from 1.03% to 1.15% for inter-day precision studies respectively, confirmed that the method was sufficiently precise.

Table: 3 Intra-day and inter-day precision studies.

Concentration µg/ml	Intra-day precision		Inter-day precision
Concentration µg/ml	Mean	% RSD	Mean	%RSD
10	2318372.68	1.44	2318667.16	1.08
20	4558859.18	1.12	4587800.13	1.03
30	6674185.59	0.98	6710632.95	1.15

System suitability:

To know reproducibility of the method, system suitability test was carried out to determine the chromatographic parameter such as theoretical plats, tailing factor, %RSD by analyzing sample solution. The results obtained in system suitability study are shown in table 4.

Table: 4 System suitability parameter of Brimonidine Tartrate.

Parameter	Brimonidine tartrate
Tailing factor	1.208
Theoretical plats	17026.75
%RSD	1.74

Accuracy:

The accuracy was expressed in term of recovery. In accuracy the recovery studies was carried out by adding known amount drug with preanalyse sample solution and content was reanalyzed by the proposed method. The accuracy was carried out by injecting the Brimonidine tartrate solution three times at three different concentration solution of 80%, 100%, 120% of the active ingredient by adding known amount of brimonidine tartrate standard to sample of known concentration. As shown in Table 5, the percent recovery ranged between 99.71% to 103.87%.

Table: 5 Recovery studies of Brimonidine Tartrate

Concentration µg/ml	Amount added (mg)	Amount found (mg)	% Recovery	% RSD
10	8	8.31	103.87	1.56
10	10	9.88	99.71	1.46
10	12	12.28	102.37	1.67

Limit of detection and limit of quantification:

The LOD and LOQ were calculated with standard deviation of response and slope of calibration curve. The LOD and LOQ for brimonidine tartrate were found to be 1.03µg mL^-1 and 3.13µg mL^-1, respectively.

Robustness:

In robustness, method was checked by changing the chromatographic condition like flow rate (+0.1 and -0.1ml/min) and absorbance wavelength (+5 and -5nm). After changes it was observed that the peak symmetry and peak response was found to be adequate. The data obtained in robustness study are shown in Table 6, indicating that the test method was robust in variable condition.

Table: 6 Robustness parameter of Brimonidine Tartrate

Parameters changed (n=5)	Flow rate(1ml/min) 0.9ml/min 1.1ml/min		Wavelength(246nm) +2nm -2nm
Mean area	23083 16.14	23485 11.18	23125 90.16	23103 44.51
SD	42869.91	31766.69	34548.91	38832.32
%RSD	1.86	1.35	1.49	1.68

CONCLUSION:

The developed method was found to be rapid, accurate, precise and reproducible. The method was linear over a wide concentration range, economical and utilizes a mobile phase which can be easily prepared. All these factors make this method suitable for the estimation of brimonidine tartrate. The developed method can be used for the routine analysis and assay of brimonidine tartrate in quality control laboratories.

REFERENCES:

1. http://drugspedia.net/prep/27554.html.[Acessed on 2 Apr 2011]

2. Mahajan A, Athensia F, Gandhi SV, Deshpande PB. Development and validation of high performance thin layer chromatographic method for estimation of Brimonidine tartrate as bulk drug and in ophthalmic solution. J Pharm. Tech. Res, 2010; 2(3); 1376-1379.

3. Shirke RR, Pai N. RP-HPLC determination of Brimonidine Tartrate in Brimonidine tartrate eye drops. Indian Drugs, 2002, 39, 484-486.

4. Madhavi A, Naidu A, Subba Rao DV, Srinivasu P. Development and Validation of a New LC Method for Analysis of Brimonidine Tartrate and Related Compounds. Chromatographia, 2009; 69; 1- 7.

5. Karamanos NK, Lamari F, Katsimpris J, Gartaganis, S. Development of an hplc method for determining the alpha2-adrenergic receptor agonist brimonidine in blood serum and aqueous humor of the eye. Biomed. Chromatogr, 1999; 13; 86- 88.

6. Jiang S, Chappa AK, Proksch JW. A rapid and sensitive LC/MS/MS assay for the quantitation of brimonidine in ocular fluids and tissues. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci, 2009; 877; 107-114.

7. Sonanis MC, Rajput AP. Development and validation of a new stability indicating analytical method for the determination of related components of Brimonidine Tartrate In drug substances and drug product using UPLC. J. Pharm. Pharma. Sci. 2011; 3; 1-6.

8. ICH, Validation of Analytical Procedures: Text and Methodology, Q2(R1), in: International Conference on Harmonization, IFPMA, Geneva, 2005.

Received on 17.04.2011 Modified on 30.04.2011

Asian J. Research Chem. 4(10): Oct., 2011; Page 1591-1593